Biology subjects

Toyofuku, M.

Publications and source records attributed to Toyofuku, M..

4 recordsLinked to original sources

bioRxiv2025

Challenging the Diffusion Barrier Paradigm: Biofilms Promote Flavin-mediated Electron Shuttling in Shewanella oneidensis

Flavins are ubiquitous diffusible redox mediators that are crucial in enabling extracellular electron transfer (EET) in electroactive bacteria that often form biofilms in both natural and engineered environments. However, the behavior of flavin diffusion within biofilms remains poorly understood. In this study, we developed a colony-based electrochemical platform using interdigitated electrode arrays to quantify the diffusion coefficients of flavins within bacterial biofilms. We found that flavin diffusivity was about 11-fold enhanced in Shewanella oneidensis MR-1 biofilms than in the bulk solution. However, this enhancement was abolished in the gene deletion mutant that lacked the membrane-associated flavin-binding c-type cytochrome OmcA, which suggests that the flavin-OmcA interaction on cell surfaces facilitates flavin diffusion within the biofilm. Notably, the diffusion coefficients of other redox molecules, such as methylene blue and safranin, scarcely improved in biofilms, which validates the inference that flavin-specific interaction-accelerated diffusion occurs in biofilm environments. These findings uncover a phenomenon promoting long-range electron transfer, overturning the prevailing assumption that shuttling-based EET is hindered by slow molecular diffusion in biofilms. Our study highlights the functional importance of cell-surface cytochromes in overcoming the kinetic limitation of diffusion-based electron transfer, thereby shaping the bioenergetics in biofilms.

microbiology↗

bioRxiv2025

A phage-derived reconfigurable effector associated with an actinobacterial contractile nanomachine tailors bacterial responses to competition

Contractile injection systems (CISs) are derivatives of phage tails and widely distributed in prokaryotes. CISs load cognate effectors and eject them through contractile actions resembling those of phage tails. Ejected effectors play central roles in CIS functionality by acting on target cells and mediating various biological processes. Here, we report a novel group of CIS effectors related to phage tapemeasure protein, the transmembrane component of the phage infection machinery. This group is broadly distributed within the class actinobacteria, one of the bacterial classes in which CIS gene clusters are highly conserved, and is represented by Sle1, a cognate effector of the intracellularly localised Streptomyces lividans phage tail-like nanoparticle (SLP). This effector is associated with Sle2, which contains a CIS effector core domain and interacts with the SLP core component. Sle1 is packaged inside SLP and is translocated to lipid membranes along with SLPs. The functional domain of Sle1, probably through interactions with ribosome-containing subcellular fractions, upregulates the membrane-associated proteome in S. lividans and E. coli. This effect modifies the physiological properties of S. lividans, ultimately enhancing its adaptation to microbial competition. In addition, we revealed that Sle1-type effectors conserved among actinobacterial species are structurally and functionally diverse in their functional domains. One of them from Micromonospora eburnea constitutes a novel toxin-antitoxin system and introducing its functional domain into Sle1 reprogrammes the phenotypic responsiveness of S. lividans to neighbouring bacteria. Our findings illustrate that phage elements can be incorporated into CISs as reconfigurable platforms for bacterial adaptation to various environmental conditions. ImportanceBacterial CISs have attracted interests for their importance in microbial ecology and potential in biotechnological applications. However, understanding of their functional diversity is currently limited because many CIS effectors remain unannotated due to a lack of inferable structural and genetic signatures. Our findings on Sle1 and its relatives illuminate a previously unidentified class of CIS effectors with phage tapemeasure protein-related modular architecture, association with the CIS effector core domain, and wide distribution within the major class of actinobacteria, substantially expanding the known repertoire of effector classes. The impact of Sle1 on S. lividans suggest a link between CIS effectors and bacterial adaptation to environmental conditions, highlighting unexplored functional diversity of CIS effectors as tuners of bacterial phenotypes in communities. This work offers routes to manipulate ecological behaviours of actinobacterial species and to access their cryptic traits through effector modulation.

microbiology↗

bioRxiv2025

Membrane vesicles of Shewanella oneidensis MR-1 enhance denitrification growth in a species-selective manner

Denitrification, a fundamental bacterial respiratory process that occurs in anoxic environments, plays a pivotal role in energy synthesis and the global nitrogen cycle. Although the significance of this process is well-recognized, its regulation within polymicrobial communities remains poorly understood, particularly concerning interspecies interactions. In this study, we investigated the role that bacterial membrane vesicles (MV) play in modulating denitrification across bacterial species. MV is known to carry specific biomolecules such as secondary metabolites, proteins, and nucleic acids, therefore considered to be a secretion pathway. We found that MV produced by Shewanella oneidensis enhanced denitrification in a species-specific manner. Bacteria with highly hydrophobic surfaces tended to respond to denitrification enhancement, suggesting that the MV-bacteria attachment process is the key to generating species specificity. Transcriptome analysis and isotopic metabolite tracking indicated that the MV influenced denitrifying activities, rather than the transcription of denitrification-related genes. We further demonstrated that c-type cytochromes in MV act as key components that enhance denitrification. These insights expand our understanding of bacterial ecology, highlighting the role of membrane vesicles in facilitating respiratory competition and cooperation in polymicrobial communities.

microbiology↗

bioRxiv2024

Droplet Sequencing Reveals Virulence Gene Clusters in Oral Biofilm Extracellular Vesicles

Bacterial extracellular vesicles (BEVs), produced by a broad spectrum of bacteria, play a crucial role in enhancing intercellular communication through DNA transfer. A vital determinant of their gene transfer efficiency is the gene content diversity within BEVs, an aspect that conventional metagenomics fails to capture. Our study bridges this gap with a novel microdroplet-based sequencing technique that precisely details DNA content within hundreds of individual BEVs. This technique revealed a unique DNA profile in BEVs from the oral pathogen Porphyromonas gingivalis, pinpointing specific genomic regions related to DNA integration (e.g., DNA transposition and CRISPR-Cas systems). These enriched genes, overlooked by standard analyses that aggregate total read counts, indicate that our method offers a more focused view into the genetic contents of BEVs. Applying our technique to dental plaque-derived BEVs, we discovered a hundredfold higher prevalence of DNA encapsulation than previously estimated, with over 30% of BEVs containing DNA. Specifically, we identified a substantial presence of O-antigen biosynthesis genes, prominent hotspots of frequent horizontal gene transfer, from Alcaligenes faecalis. Given that O-antigens mediate host-bacterial interactions, this gene enrichment in the large fraction of BEVs suggests a potential novel pathway by which BEVs could influence pathogenicity within oral biofilms. Our research unveils critical insights into the potential functions of vesicular DNA in microbial communities, establishing a powerful platform for studying vesicular DNA in microbiomes. This technical breakthrough provides a foundational basis for future research in microbial communication and the development of potential therapeutic or diagnosis strategies. Significance StatementBEVs have been studied for decades, yet their roles in nature and disease are just beginning to be appreciated. Our study makes a significant leap in understanding the roles of BEVs as gene transfer vehicles. By developing a microdroplet-based sequencing technique, we have uncovered detailed DNA profiles within individual BEVs, a task beyond the capabilities of conventional metagenomic methods. This breakthrough highlights specific genomic regions enriched in BEVs from pure culture and human dental plaque. Furthermore, the high prevalence of biofilm BEVs enriched in O-antigen biosynthesis genes, suggests a potential impact on the pathogenicity of oral biofilms. This research establishes a new methodological platform for exploring the intricacies of BEV-mediated interactions in a complex microbial community.

microbiology↗